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Re: [ccp4bb] protein degradation?
 

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CCP4bb <-- 2007 <-- November 2007 <-- 04 November 2007
Previous message:
Subject: Re: protein degradation?
From: Vincent Chaptal vchaptal {- at -} MEDNET {- dot -} UCLA {- dot -} EDU
Date: 2007-11-04 		Next message:
Subject: Re: protein degradation?
From: Artem Evdokimov artem {- at -} XTALS {- dot -} ORG
Date: 2007-11-04


Subject: Re: protein degradation?
From: price {- at -} UCHICAGO {- dot -} EDU price {- at -} UCHICAGO {- dot -} EDU
Date: 2007-11-04
Some proteases are metal-dependent, and inhibitors for those aren't
Ni-column-compatible. "We" (meaning my students) found that it helps
to (1) work very quickly and (2) put EDTA into the fraction collector
tubes before eluting from the Ni column.


At 06:22 AM 11/4/2007, Vijay Kumar wrote:
>Hi,
>
>I have been trying purify a N-ter his-tagged protein over-expressed
>in E.coli. After purification (Ni-NTA or Co-Talon), I find two bands
>in SDS PAGE which are very close each other (top band in the right
>MW and more intense than the lower band). Western blot (for his-tag)
>of the gel gave signal for both the bands. Mass spec results
>confirmed both protein bands are the same. So I think it could be
>C-ter degradation of my protein. Also the 2 bands exist after
>ion-exchange and sizing column.
>
>I use commercially available complete protease inhibitor tablets
>(increasing concentration has no effect) and sonication for lysis. I
>am wondering if people have encountered the same problem and got any
>suggestions?
>
>
>Thanks in advance.
>
>Regards,
>
>Vijay

---------------------------------------------------------------------------------------------------------------------------
Phoebe A. Rice
Assoc. Prof., Dept. of Biochemistry & Molecular Biology
The University of Chicago
phone 773 834 1723
fax 773 702 0439
http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123
http://www.nasa.gov/mission_pages/cassini/multimedia/pia06064.html

CCP4bb navigation

CCP4bb <-- 2007 <-- November 2007 <-- 04 November 2007
Previous message:
Subject: Re: protein degradation?
From: Vincent Chaptal vchaptal {- at -} MEDNET {- dot -} UCLA {- dot -} EDU
Date: 2007-11-04 		Next message:
Subject: Re: protein degradation?
From: Artem Evdokimov artem {- at -} XTALS {- dot -} ORG
Date: 2007-11-04


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